Cholecalciferol (vitamin D.sub.3) is hydroxylated in the liver to 25-hydroxycholecalciferol. This metabolite, which, like the parent vitamin, promotes bone calcium mobilization and stimulates intestinal calcium transport, and serves as a precursor for the physiologically active form, 1,25-dihydroxycholecalciferol, of the vitamin is present in relatively low levels in the circulatory system. See J. L. Omdahl and H. F. DeLuca, Physiological Reviews, 53, 327 (1973) and H. K. Schnoes and H. F. DeLuca, Vitamins and Hormones, 32, 385 (1974). To detect these relatively low blood levels of 25-hydroxycholecalciferol by radioimmunological techniques requires the tritium labelling of the metabolite in very high specific activity. See, for example, W. T. Newton and B. M. Jaffe in "Radioassay In Clinical Medicine", W. T. Newton and R. M Donati, ed., Charles C. Thomas, Springfield, Ill., 1974, pages 13 to 17.
The desired labelling of 25-hydroxycholecalciferol has been accomplished by two reported procedures (T. Suda et al., Analyt. Biochem., 43, 139 [1971] and P. A. Bell and W. P. Scott, J. Labelled Compounds, 9, 339 [1973]). The first involves the addition of tritio-methyl Grignard to 25-keto-7-dehydrocholesterol (I) followed by photolysis and thermolysis of the intermediate provitamin II to yield 25-hydroxycholecalciferol-26(27)-.sup.3 H (III). The second involves the addition of the same Grignard reagent to 25-ketocholecalciferol (IV) to afford directly the labelled metabolite III. As noted, both prior processes utilize tritio-methyl Grignard reagent, prepared from formaldehyde and sodium borotritide having one atom of tritium per mole of Grignard reagent to introduce the label. As such, the introduction of the label is limited to one tritium atom per molecule of 25-hydroxycholecalciferol, resulting in the labelled metabolite having a specific activity of up to about 10 Ci/mmole. Thus, it is apparent that to achieve the labelled metabolite with the requisite high specific activity would require multiple labelling with fully enriched tritium, i.e., the addition of molecular tritium to an unsaturated system prior to elaboration of the extended olefinic system of the metabolite. ##STR1##
The present invention relates to 25-hydroxycholecalciferol-23,24-.sup.3 H having the requisite high specific activity and to a novel efficient process for the preparation thereof from readily available precursors involving the addition of two molecules of tritium to an acetylenic linkage. More particularly, the present process aspect relates to a method of synthesizing 25-hydroxycholecalciferol-23,24-.sup.3 H comprising the steps of rearranging 3.beta.,25-dihydroxy-3.alpha.,5-cyclo-5.alpha.-cholest-23-yne, in which the hydroxy groups may be conventionally protected by ether moieties, to 3.beta.,25-dihydroxycholest-5-en-23-yne 3-acylate, acylating 3.beta.,25-dihydroxy-cholest-5-en-23-yne 3-acylate to 3.beta.,25dihydroxy-cholest-5-en-23-yne. 3.beta.,25-diacylate, tritiating 3.beta.,25-dihydroxycholest-5-en-23-yne 3.beta.,25-diacylate to 25-hydroxycholesterol-23,24-.sup.3 H diacylate, converting 25-hydroxycholesterol-23,24-.sup.3 H diacylate to 25-hydroxy-7-dehydrocholesterol-23,24-.sup.3 H diacylate, hydrolyzing 25-hydroxy-7-dehydrocholesterol-23,24-.sup.3 H diacylate to 25-hydroxy-7-dehydrocholesterol-23,24-.sup.3 H, irradiating 25-hydroxy-7-dehydrocholesterol-23,24 -.sup.3 H to 25-hydroxyprecholecalciferol-23,24-.sup.3 H and isomerizing 25-hydroxyprecholecalciferol-23,24-.sup.3 H to 25-hydroxycholecalciferol-23,24-.sup.3 H.
In the formulas presented herein, the various substituents are illustrated as joined to the steroid and cholecalciferol nuclei by one of three notations: a solid line (--) indicating a substituent which is in the 62 -orientation (i.e., above the plane of the molecule), a dotted line (- - -) indicating a substituent which is in the .alpha.-orientation (i.e., below the plane of the molecule), or a wiggly line ( ) indicating a substituent which may be in the .alpha.- or .beta.-orientation or may be a mixture of both forms. The formulas have all been drawn to show the compounds in their absolute sterochemical configurations. Since the starting materials are derived from naturally occurring materials, the final products exist in the single absolute configuration depicted herein. However, the processes of the present invention are intended to apply as well to the synthesis of steroids of the racemic series. Thus, one may begin the synthesis utilizing racemic starting materials to prepare racemic products. Optically active products can then be prepared by resolution of the racemic products utilized in the preparation thereof, as hereinafter described, by standard resolution techniques well-known in the art.
As used throughout the specification and the appended claims, the term "alkyl group" refers to a monovalent substituent consisting solely of carbon and hydrogen of from 1 to 20 carbon atoms which may be straight or branched-chain. Examples of alkyl groups are methyl, ethyl, n-propyl, i-propyl, tert-butyl, hexyl, octyl and so forth. The term "alkylene group" refers to a divalent substituent consisting solely of carbon and hydrogen of from 1 to 20 carbon atoms which may be straight or branched-chain and whose free valences are attached to two distinct groups. Examples of alkylene groups are methylene, ethylene, propylene, butylene and so forth. The term "alkoxy group" refers to a monvalent substituent which consists of an alkyl group linked through an ether oxygen having its free valence bond from the ether oxygen. Examples of alkoxy groups are methoxy, ethoxy, isopropoxy, tert-butoxy and so forth. The term "phenyl alkoxy" refers to an alkoxy group which is substituted by a phenyl ring. Examples of phenyl alkoxy groups are benzyloxy, 2-phenylethoxy, 4-phenylbutoxy and so forth. The term "alkanoyloxy group" refers to the residue of an alkylcarboxylic, an alkanoic acid, formed by removal of the hydrogen from the hydroxyl portion of the carboxyl group. Examples of alkanoyloxy groups are formyloxy, acetoxy, butyryloxy, hexanoyloxy and so forth. The term "alkanoyl group" refers to the residue of an alkylcarboxylic acid, an alkanoic acid, formed by removal of the hydroxy group from the carboxylic acid moiety. Examples of alkanoyl groups are formyl, acetyl, butyryl, hexanoyl and so forth. The term "lower" as applied to any of the aforementioned groups refers to those groups having from 1 to 8 carbon atoms. The Greek letter xi (.xi.) in the name of a cholecalciferol intermediate indicates that the stereochemistry of the substituent to which it refers is undefined.